Several techniques are known for cloning genes. In the mean time, the difficulties met in the attempts to assign a specific function to the cloned genes, are known as well. On the other hand, knowing the genes which compose human DNA is the basic step which has to be coped with, if one just wishes to hope to be able to diagnose at the right time and hence properly treat a very large number of diseases, which, still to date, are regarded as hopeless.
Human genes are very numerous (they are thought to be in a number of the order of 100,000) and each of them performs a particular task: from one gene the colour of the eyes, and from another gene the blood group depend; from the genes also the hereditary diseases depend, as well as the arising and transmission of certain tumoral forms.
Identifying the individual genes is therefore of interest for the biologist, the physician, and all those who from such a knowledge should draw, as said, suggestions for diagnoses and treatment therapies with the preparation of the relevant kits and medicines.
It is well-known that the nucleotidic sequence can be used to predict which protein a cloned gene will encode, and can suggest the class to which the protein could belong, but, in general, the problem of the identification of the function of a cloned gene is still unsolved and to date, in spite of studies carried out in depth worldwide, only a few tens of human genes were identified and isolated.
The present Applicant has found now a method for identifying and/or isolating genes which code for membrane receptors of whatever biological origin, which is much simpler and faster than the analogous methods based on the expression of receptors in eucaryotic cells, and which is extremely useful when addressed to the cloning of the membrane receptors.
The conventional methods for cloning receptors for neurotransmitters require the purification of the receptor, the generation of partial sequences of aminoacids and the use of synthetic oligonucleotides to be used as probes to obtain cDNA or genomic clones: however, in the case of most neurotransmitter receptors, their rarity renders them extremely difficult to purify, and only very seldom has the cloning of receptors by expression been carried out successfully [Masu Y., Nakayama K., Tamaki H., Harada Y., Kuno M., Nakahishi S. (1987) Nature 329 836-838; Julius D., MacDermott A. B., Axel R., Jessel T. M. (1988) Science 241 558-564; Yokota Y., Sasai Y, Tonaka K., Fujiwara T., Tsuchida K., Shigemoto R., Kakizuka A., Okhubo H., Nakanishi S. (1989) J. Biol. Chem. 264 17649-17652; Shigemoto R., Yokota Y., Tsuchida K., Nakanishi S. (1990) J. Biol. Chem. 265 623-628; Tanaka K., Masu M., Nakanishi S. (1990) Neuron 4 843-854].
Functional receptor molecules are obtained as well by microinjecting in vitro synthetized mRNA into oocites of Xenopus, and also some marginal indications exist on the functional expression in E. coli of some genes for human receptors [Marullo S., Delavier-Klutchko C., Eshdat Y., Strosberg A. D., Emorine L. (1988) Proc. Natl. Acad. U.S.A. 85 7751-7555]. Unfortunately, none of these reports makes possible a practical technique for identifying and isolating genes endowed with a specific receptor activity to be developed.
The method according to the present invention makes it possible to overcome these limitations, which, to date, have to be ascribed to the technologies known from the prior art and, exploiting the above cited possibility of functional expression of the receptors in E. coli and their interaction with specific ligands, makes it possible to identify recombinant clones coding for the genes of said receptors. The present method essentially consists in the replication on filters of the bacterial colonies expressing membrane receptors and their labeling with radioactive ligands.